Mycobacteria have long been recognized as bacterial pathogens of man and continue to produce devastating illness, particularly in developing countries. World Health Organization, Bull. WHO, 61, 779 (1983). Tuberculosis is caused by respiratory infection with Mycobacterium tuberculosis (M. tuberculosis) and currently afflicts about 30 million people worldwide with an annual mortality of about 3 million.
Crude bacterial antigen preparations have been used for immunodiagnostic and immunoprophylactic purposes. The tuberculin test developed by Koch in 1881 was the first immunodiagnostic test used in man. Tuberculin, an M. tuberculosis filtrate of complex but poorly defined composition, is currently used as a delayed-type cutaneous hypersensitivity (DCH) or skin test antigen to detect prior exposure to the pathogen. Seibert et al., Am. Rev. Tuberc., 69, 585 (1954). Unfortunately, the utility of tuberculin is limited both by its lack of specificity and by its inability to distinguish between active disease, prior sensitization by contact with M. tuberculosis or cross-sensitization to other mycobacteria.
Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis (M. bovis), is currently used as a live vaccine to protect against tuberculosis in man. Calmette, A., J. Am. Med. Assoc., 96, 58 (1931). While BCG has been effective in reducing the incidence of tuberculosis in Western Europe [Medical Research Council, Bull. WHO, 46, 371 (1972)], it has recently been found to be ineffective in a major trial in India [World Health Organization, WHO Tech. Rep. Ser., 651 (1980)].
A basic problem with the crude antigen preparations that are now used to detect mycobacterial infections is that the antigen preparations often react positively with several different mycobacterial species. This, of course, complicates diagnosis and the selection of an appropriate treatment regimen. A reagent that specifically evokes an immune response against a particular mycobacterial species would be beneficial in the diagnosis and management of mycobacterial infections.
The advantages of the use of a defined polypeptide antigen in a DCH reaction are numerous. For example, in the case of mycobacterial infections, since the amino acid residue sequence of the polypeptide corresponds to a portion of a protein that is specifically expressed in the tuberculous mycobacterial species, the polypeptide may be a useful reagent for specifically detecting a tuberculous mycobacterial infection and thereby circumventing the cross-reactivity problems associated with the currently-used skin test antigens. Moreover, the polypeptide may be chemically synthesized to eliminate the need to grow large cultures of a pathogenic organism for the production of skin test antigens.
The polypeptide may also be used in the detection or prevention (vaccination) of mycobacterial infections. In fact, an inoculum containing the polypeptide could replace tuberculin or PPD (purified protein derivative) as the antigen of choice in DCH or skin tests for the detection of tuberculosis in humans.
While the general concept of preparing synthetic antigens (immunogens) and using them to induce antibodies of predetermined specificity has been described, there remains a large area of this technology that continues to defy predictability. There are at least two reasons for this. First, a synthetic antigen (immunogen) does not necessarily induce antibodies that immunoreact with the intact protein in its native environment. Second, the natural antibodies of a host to a naturally occurring immunogen, such as a viral protein, rarely immunoreact with a polypeptide that corresponds to a short linear portion of the immunogen's amino acid residue sequence. This latter phenomenon is believed to be the result of short linear polypeptides lacking required secondary and tertiary conformational structures.
Much of the work on the binding of peptide by antibody made to proteins is summarized in a review by Benjamini, E., et al., Current Topics in Microbiology and Immunology, 58, 85 (1972). The role of peptide structure in antibody binding has been emphasized by Goodman, J. W., Immunochem 6, 139 (1969).
Most of the studies that involve the effects of changes in the sequence of peptides on antibody binding have been interpreted as indicating that the structure of the antibody combining site plays a predominant role. The effect of sequence and structural changes in these studies is intermixed and difficult to segregate. Some of these studies can equally well be explained by structural changes in antigen effecting the binding.
Antibody response at the molecular level involves binding of an antigen of defined sequence (primary structure) and in a defined conformation (secondary and tertiary structure). Immune response to protein antigens has traditionally been interpreted as being directed against primary, secondary or tertiary structue of the protein.
This classification scheme may have some validity for proteins that have a well defined overall structure at physiological temperatures and solutions. However, its validity is in doubt for peptide antigens that have a more dynamic structure.